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Two interconverting Fe(IV) intermediates in aliphatic chlorination by the halogenase CytC3

Nature Chemical Biology volume 3, pages 113–116 (2007)Cite this article

Abstract

Enzymatic incorporation of a halogen atom is a common feature in the biosyntheses of more than 4,500 natural products1,2,3,4,5. Halogenation of unactivated carbon centers in the biosyntheses of several compounds of nonribosomal peptide origin is carried out by a class of mononuclear nonheme iron enzymes that require α-ketoglutarate (αKG, 1), chloride and oxygen6. To investigate the ability of these enzymes to functionalize unactivated methyl groups, we characterized the chlorination of the γ-methyl substituent of L-2-aminobutyric acid (L-Aba, 2) attached to the carrier protein CytC2 by iron halogenase (CytC3) from soil Streptomyces sp. We identified an intermediate state comprising two high-spin Fe(IV) complexes in rapid equilibrium. At least one of the Fe(IV) complexes abstracts hydrogen from the substrate. The demonstration that chlorination proceeds through an Fe(IV) intermediate that cleaves a C-H bond reveals the mechanistic similarity of aliphatic halogenases to the iron- and αKG-dependent hydroxylases.

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Figure 1: Absorption spectroscopy of CytC3.
Figure 2: Mössbauer spectroscopy of CytC3.

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Acknowledgements

E.R. Strieter is gratefully acknowledged for a generous gift of (R,R)-(−)-pseudoephedrine glycinamide hydrate. We thank F.H. Vaillancourt and J.A. Read for careful proofreading of the manuscript. This work was supported by the US National Institutes of Health (NIH GM-69657 to J.M.B. and C.K.; GM-20011 and GM-49338 to C.T.W.), the donors of the American Chemical Society Petroleum Research Fund (ACS-PRF 41170-G3 to C.K.), the Arnold and Mabel Beckman Foundation (Young Investigator Award to C.K.) and the Dreyfus Foundation (Camille Dreyfus Teacher Scholar Award to C.K.). D.P.G. is supported by the Damon Runyon Cancer Research Foundation Postdoctoral Fellowship (DRG-1893-05).

Author information

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, 02115, Massachusetts, USA

    Danica P Galonić & Christopher T Walsh

  2. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, 16802, Pennsylvania, USA

    Eric W Barr, J Martin Bollinger Jr & Carsten Krebs

  3. Department of Chemistry, The Pennsylvania State University, University Park, 16802, Pennsylvania, USA

    J Martin Bollinger Jr & Carsten Krebs

Authors
  1. Danica P Galonić
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  2. Eric W Barr
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  3. Christopher T Walsh
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  4. J Martin Bollinger Jr
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  5. Carsten Krebs
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Contributions

D.P.G., C.T.W., J.M.B. and C.K. designed the experiments and wrote the manuscript. D.P.G. overproduced and purified proteins, synthesized deuterated substrate, and performed iron titration and SF absorption experiments. D.P.G. and E.W.B. performed FQ experiments. C.K. collected and analyzed Mössbauer spectra.

Corresponding authors

Correspondence to Christopher T Walsh, J Martin Bollinger Jr or Carsten Krebs.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Comparison of the 4.2 K/zero-field Mössbauer reference spectrum of the Fe(IV) intermediates as described in the main manuscript (solid lines) to reference spectra of the Fe(IV) intermediates of samples prepared under modified reaction conditions. (PDF 108 kb)

Supplementary Scheme 1

Enzymatic conversion of L-Aba to γ-Cl-L-Aba in cytotrienin-producing Streptomyces sp. (PDF 66 kb)

Supplementary Methods (PDF 46 kb)

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Galonić, D., Barr, E., Walsh, C. et al. Two interconverting Fe(IV) intermediates in aliphatic chlorination by the halogenase CytC3. Nat Chem Biol 3, 113–116 (2007). https://doi.org/10.1038/nchembio856

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